At the present time, treatments for most genetic diseases do little to alleviate the symptoms associated with the genetic disease and considerable effort is currently underway to develop new, safe and effective methods of treatment. Recent progress in the areas of molecular biology and genetic engineering have lead to the isolation and characterization of genes associated with genetic diseases. This in turn has lead to the development of the concept of gene therapy i.e., the replacement or supplement of defective genetic information with normal functional genes, and its potential use for treatment of genetic disorders.
The most well studied models for gene therapy involve gene transfer using recombinant pathogenic viruses to express new genetic information in order to correct disease phenotypes. Until recently, the most widely researched viral vectors for use in gene therapy were the retroviruses (Miller, A. D., 1990, Human Gene Ther. 1:5-14). One problem associated with retroviral use is the random integration of retroviruses into the host genome which can lead to insertional mutagenesis. In addition, the long terminal repeats (LTR) structures located at the ends of the retroviral genome contain promoter/enhancer activity that may lead to activation of genetic loci located adjacent to the integrated viral DNA. For example, integration of retroviral DNA adjacent to a proto-oncogene may lead to inadvertent activation of proto-oncogene expression which may, in turn, lead to transformation and tumorigenesis. This is illustrated, for example, by recent evidence indicating that retrovirus vectors in non-human primates results in T cell lymphomas. More recent efforts in the field of gene therapy have focussed on the development of viral vectors lacking the deleterious characteristics of the retroviruses.
In addition to the retroviruses, the adeno associated viruses have also been studied as an alternative system for delivery of stable genetic information into the cell. The AAV genome is composed of a linear single stranded DNA molecule of 4680 nucleotides which contains major open reading frames coding for the Rep (replication) and cap (capsid) proteins. Flanking the AAV coding regions are the two 145 nucleotide inverted terminal (ITR) repeat sequences that contain palindromic sequences that can fold over to form hairpin structures which function as primers during initiation of DNA replication (FIG. 1). In addition, the ITR sequences are needed in cis, for viral integration, rescue from the host genome and encapsidation of viral genomic DNA, into mature virions (Muzyczka, N. 1992, Current Topics in Microbiology & Immunology. 158, 97-129).
AAV can assume one of two pathways upon infection into the host cell. In the presence of helper virus, AAV will enter the lytic cycle whereby the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome integrates as a provirus into of the host cell genome through recombination between the AAV termini and host cell sequences (Cheung, A. et al., 1980, J. Virol. 33:739-748; Berns, K. I. et al., 1982, in Virus Persistence, eds. Mahey, B. W. J., et al. (Cambridge Univ. Press, Cambridge), pp. 249-265).
Characterization of the proviral integration site and analysis of flanking cellular sequences indicates that AAV viral DNA integrates specifically into the long arm of human chromosome 19 (Kotin, R. M. et al., 1990, Proc. Natl. Acad. Sci. USA 87:2211-2215; Samulski, R. J. et al., 1991, EMBO J. 10:3941-3950). This particular feature of AAV reduces the likelihood of insertional mutagenesis resulting from random integration of viral vector DNA into the coding region of a host gene. Furthermore, in contrast to the retroviral LTR sequences, the AAV ITR (inverted terminal repeat) sequences appear to be devoid of transcriptional regulatory elements, reducing the risk of insertional activation of proto oncogenes.
Recent work with AAV has been facilitated by the discovery that AAV sequences cloned into prokaryotic vectors are infectious (Samulski, et al. 1982, Proc. Natl. Acad. Sci. U.S.A. 79:2077-2081). For example, when a plasmid containing an intact AAV genome is transfected into cells in the presence of helper virus, AAV can be rescued out from the plasmid vector and enter the lytic pathway leading to production of mature virions. In the absence of helper virus the recombinant AAV vector will integrate into the host cell genome and remain as a provirus until the cell subsequently becomes infected with a helper virus.
One problem associated with the use of vectors containing an intact AAV genome is the size limitations imposed on the vectors by the packaging of DNA fragments into mature viral particles. Prior to the present invention it was unknown what viral sequences were required for replication, and encapsidation of the DNA into viral particles, or alternatively, for integration of vector DNA into the host genome. The present invention defines a novel 165 bp sequences derived from AAV that can function to direct the replication and encapsidation of DNA into viral particles and/or the integration of vector DNA into the targeted host genome.